SMRT Sequencing Enables Discovery of Epigenetic Driver of C. diff Persistence

How do pernicious pathogens like Clostridioides difficile spread through hospitals and persist so tenaciously in the human gut, leading to about half a million infections and 30,000 deaths each year? 
It’s a mystery scientists have been anxious to solve, and they’ve invested countless hours of research into the bacteria’s physiology, genetics and genomic evolution.  
A team from Mount Sinai School of Medicine in New York City has uncovered an important new clue by studying an overlooked aspect of C. difficile’s biology: Epigenetics. 
Using PacBio SMRT Sequencing and comparative epigenomics, Pedro H. Oliveira (@pholive81), Gang Fang (@iamfanggang), and colleagues mapped and characterized the DNA methylomes of 36 human C. difficile isolates. 
As described in a recent Nature Microbiology paper, while they observed substantial epigenomic diversity across C. difficile isolates, they noticed one methyltransferase (MTase) was highly conserved across all of the isolates (and, they later discovered, in another ~300 published C. difficile genomes). This MTase, which they dubbed camA, shared a common methylation motif — CAAAAA, with the last adenine methylated at the N6 position, namely 6mA. 
“Despite the small sample size, I got excited wondering if this methylation pattern might be conserved in this critical pathogen and play important roles in regulating its physiology,” Fang wrote in a Behind The Paper feature.
That left the question, how does it work? The Mount Sinai team reached out to other experts in the field, Aimee Shen at Tufts University and Rita Tamayo at the University of North Carolina, to do some in vitro and in vivo studies. 
They found that inactivation of the gene encoding this MTase compromises spore formation, a key step in both the transmission of C. difficile and its ability to persist in the intestinal tract. 
“Further experimental and integrative transcriptomic analysis suggested that epigenetic regulation by DNA methylation also modulates the cell length, host colonization and biofilm formation of C. difficile,” the authors wrote. 
The discovery could have a direct translational impact. The fact that camA is conserved across all of the C. difficile genomes but is present in just a few Clostridiales makes it a promising, highly specific drug target. Furthermore, as the MTase does not seem to impact the general fitness of C. difficile, a drug that specifically targets it might also have a lower chance for resistance.
“These findings provide a unique epigenetic dimension to characterize medically relevant biological processes in this important pathogen,” the authors concluded.
The discovery was made as part of the Mount Sinai Pathogen Surveillance Program, which we previously profiled for its innovative use of genetic sequencing in disease control. 
The authors noted that such high-resolution mapping of bacterial DNA-methylation events has only recently become possible with the advent of PacBio’s single molecule, real-time sequencing.
“This technique enabled the characterization of the first bacterial methylomes and, since then, more than 2,200 (as of September 2019) have been mapped, heralding a new era of bacterial epigenomics,” they added. 
Learn more about the methods and workflow for direct detection of epigenetics using PacBio sequencing.